靶向运载siRNA调控肾癌VEGF Pre-mRNA选择性剪接的作用及机制研究

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon alternative splicing site choice in eighth exon. Since sustained angiogenesis is critical for renal cell carcinoma growth and metastasis, restoration of normal VEGF splicing presents a new approach. Recent studies have shown that aberrant splice site targeted siRNA efficiently restored splicing without reducing transcript levels, providing a novel strategy for the regulation of aberrant gene splicing in cancer cells. In this project, we will detect the molecular basis by which the pro-angiogenic splice site choice is mediated, and then specifically modulate VEGF splicing to promote anti-angiogenic isoforms in renal cell carcinoma by splice site-based siRNA. We have previously shown that siRNA could inhibit RNA polymerase II function at transcriptional level. As RNA polymerase II has been implicated in regulating alternative splicing, we hypothesized that siRNA may interfere splicing through indirect interaction with polymerase II. Our preliminary studies also confirmed that cell-penetrating peptide (CPP) could deliver fusion protein into tumor cells through an efficient and safe way. In the project, we will design and synthesize the renal cell carcinoma targeted fusion protein of single-chain antibody against G250 and low molecular weight protamine to transport siRNA. This work will build a solid theoretical foundation for using siRNA to regulate alternative splicing, and develop a more selective strategy for targeted therapy in renal cell carcinoma.

血管内皮生长因子(VEGF) mRNA前体第8外显子选择性剪接可产生促血管生成和抗血管生成两类功能相反的变构体，靶向干预VEGF异常剪接已成为肾癌治疗研究的新内容。新近研究表明，针对可变剪接位点的siRNA能够在未沉默基因表达前提下，有效干预mRNA前体选择性剪接，为靶向调控肿瘤细胞基因异常剪接提供了新方法。据此，本课题将系统解析肾癌细胞VEGF 异常剪接的分子基础并设计siRNA进行靶向干预；在前期研究发现siRNA能够在转录水平抑制RNA聚合酶II功能的基础上，探讨siRNA干预选择性剪接的作用机制；在前期研究证实穿膜肽能够安全高效地将融合蛋白导入肿瘤细胞的基础上，设计并合成具有肾癌组织靶向性的G250单链抗体-低分子量鱼精蛋白(LMWP)融合蛋白载体运载siRNA。本项目的成功实施，将为进一步利用siRNA调控基因选择性剪接奠定理论基础，并为肾癌的靶向治疗提供更具针对性的新策略。

血管内皮生长因子(VEGF) mRNA前体第8外显子选择性剪接可产生促血管生成和抗血管生成两类功能相反的变构体，靶向干预VEGF异常剪接已成为肾癌治疗研究的新内容。针对可变剪接位点的siRNA能够在未沉默基因表达前提下，有效干预mRNA前体选择性剪接，为靶向调控肿瘤细胞基因异常剪接提供了新方法。本课题证实了促血管生成的VEGF剪接体（VEGF165a）在肾癌中表达明显上调，而抗血管生成的VEGF剪接体（VEGF165b）在肾癌中表达明显下调；针对特定剪接位点设计的靶向siRNA能够有效干预VEGF选择性剪接，使VEGF165a表达下降， VEGF165b表达显著上升；与此同时，肾癌细胞增殖、迁移、促血管生成能力显著下降；进一步研究显示，转染针对VEGF剪接位点的siRNA后，剪接位点周围结合的AGO2蛋白显著增多、剪接体蛋白组分SF2显著减少。本研究为进一步利用siRNA调控基因选择性剪接奠定理论基础，并为肾癌的靶向治疗提供更具针对性的新策略。此外，本课题研究发现miR-106b-5p能够直接作用于肾癌抑制基因SETD2 mRNA 3’UTR，进而在转录后水平抑制SETD2表达，为肾癌的发病机制研究提供了新理论。